This invention relates, inter alia, to the construction of cloning vectors that contain deoxyribonucleic acid (DNA) sequences which code for part or all of the hormone inhibin, and of host cells such as bacterial strains containing such vectors, and of host cells such as bacterial strains which produce part, all or precursors of inhibin. In addition it relates to the production and uses of expression products of said vectors and strains and to the production and uses of fragments of the expression products and vectors, be they natural or synthetic in origin.
It was suggested in 1932 that the gonads produce a non-steroidal factor called inhibin which is involved in feedback regulation of pituitary function (McCullagh, D. R. (1932) Science 76, 19-20). Since that time, it has been shown that the anterior pituitary produces at least two gonadotrophins, follicle stimulating hormone or follitropin (FSH) and luteinising hormone or lutropin (LH) which together regulate the development and functioning of the gonads. Sensitive radioimmunoassays have permitted accurate independent monitoring of each hormone and have shown feedback regulation of these hormones by the gonads. The feedback regulation of LH appears to be predominantly via steroids whereas that of FSH is via the protein or glycoprotein factor, inhibin, in addition to steroids.
Inhibin can now be defined as a protein or glycoprotein hormone secreted from the granulosa cells in the ovary or Sertoli cells in the testis. It is secreted in response to FSH and acts on the pituitary as a feedback inhibitor of FSH synthesis and secretion but which leaves the basal synthesis and secretion of LH largely intact. Whether inhibin, its mRNA or precursors are formed in other cells and tissues is not yet known.
Since the early 1970""s a number of attempts have been made to purify inhibin from a variety of gonadal sources, both testicular and ovarian, and with conflicting results (de Jong, F. H. (1979) Mol. Cell. Endocrinol. 13, 1-10) due in part to the use of a variety of bioassay systems where any suppression of FSH in pituitary cells in vitro or in vivo was assumed to be due to inhibin and checks were not always made for non-specific toxic effects of the test substances (Baker, H. W. G. et al. (1981) in Intragonadal Regulation of Reproduction (Franchimont, P. and Channing, C. P. Eds.), Academic Press, London pp. 193-228; Baker H. W. G. et al., (1982) Ann. New York Acad. Sci. 383, 329-342).
Recently, inhibin from bovine follicular fluid (bFF) has been purified to homogeneity. (International Patent Application PCT/AU85/00119; Robertson, D. M. et al., (1985) Biochem. Biophys. Res. Commun. 126, 220-226). This achievement was aided by the use of a rigorous cultured rat pituitary cell assay (Scott, R. S. et al., (1980) Endocrinol. 107, 1536-1542) which incorporates a means of assessing the cytotoxic effects of the substances under test (Robertson, D. M. et al., (1982) Mol. Cell. Endocrinol. 26, 119-127) so that non-specific toxic effects that lowered the FSH content of the cells under measurement could be identified as distinct from the effects of inhibin. The standard employed was a bovine follicular fluid preparation with an inhibit activity of 20 U/ml in terms of a previously described ovine testicular lymph standard assigned an arbitary activity of 1 U/mg (Scott, R. S. et al., (1980) Endocrinol. 107, 1536-1542)
The inhibin from bFF was purified about 3,500-fold to a specific activity of 200,000 units/mg protein and is a protein of 58 kD composed of two disulphide-linked subunits A and B of approximately 43 kD and 15 kD respectively as evidenced from electrophoresis in polyacrylamide gels in the presence of sodium dodecylsulphate (SDS-PAGE). The amino acid sequences for the NH2-terminus of each subunit have been determined. This purified substance has the in vitro physiological properties defined earlier as those of inhibin, namely inhibiting the synthesis and release of endogenous FSH from rat pituitary cells whilst leaving intact the synthesis and release of LH prolactin and thyroid stimulating hormone.
Since FSH is important for determining the incidence and rate of ovulation in females and spermatogenesis in males, it follows that the main potential applications of inhibin, analogues, or homologues of inhibin or antibodies against inhibin will be to inhibit or to stimulate gonadal function in man and domestic animals and as diagnostic tools for analysis of gonadal function. Many experiments have been performed in vivo using crude or partially fractionated gonadal extracts or secretions in attempts to analyse the physiological effects of inhibin. Effects attributed to be due to the inhibin or antibodies against inhibin in these experiments include
1. Inhibition of gonadal function (Moudgal, N. R. et al. (1985) in Gonadal Proteins and Peptides and their Biological Significance (Sairam, M. R. and Atkinson, L. E., Eds.) World Scientific Publishing Co., Singapore, pp 21-37).
2. An increase in ovulation rate (O""Shea, T. et al., (1982) Proc. Aust. Soc. Rep. Biol. 14, 85; O""Shea, T. et al. (1983) Proc. Aust. Soc. Rep. Biol. 15, 22; Henderson, K. M. et al., (1984) J. Endocrinol. 102, 305-309).
3. An advancement of the onset of puberty (Al-Obaidi, F. A. R. et al., (1983) Proc. Aust. Soc. Rep. Biol. 15, 80).
The commercial exploitation of these properties and further physiological studies in live animals require large quantities of pure inhibin or fragments thereof or inhibin agonists and antagonists whether of natural or synthetic origin. Such quantities cannot be obtained solely by the present methods of purification since source material may be limited (e.g. human follicular fluid) and a typical extraction starting with 50 ml bFF yields only 5-10 xcexcg of purified material.
The present invention seeks, inter alia, to overcome these limitations by the identification and characterisation of the genes which code inhibin so as to allow the molecular cloning of genes or parts of genes coding for inhibin into a host such as Escherichia coli and by manipulating the cloned genes or parts thereof to create hosts, such as bacterial strains, which can synthesise all, part or precursors of the inhibin molecule, including subunits thereof.
In a first embodiment the present invention provides a first DNA sequence which acts as a coding sequence for amino acid sequences of all, part of precursors of inhibin, or a DNA sequence which hybridises to said first DNA sequence, said sequences being from whatever source obtained, including natural, synthetic or semi-synthetic sources, said sequences including sequences related by mutation, including single or multiple base substitutions, deletions, insertions, and inversions and including DNA sequences which on expression code for all, part or precursors or homologues and analogues of a polypeptide which is inhibin or which displays similar immunological or biological activity as that of inhibin.
Preferred sequences of the invention are those coding for a polypeptide corresponding to the 43 kD and 15 kD (A and B) subunits of bovine and human inhibin as described hereinafter in more detail and depicted in FIGS. 5, 6, 7 and 8 of the accompanying drawings.
In another preferred embodiment, the DNA codes for the 20 kD (AC) subunit of inhibin described hereinafter.
The DNA sequences embraced by the present invention can be prepared for example from vertibrate cells by extracting total DNA therefrom and isolating the sequences by standard techniques. Alternatively the DNA may be prepared in vitro, synthetically or biosynthetically, such as by the use of an mRNA terplate.
Also, within the scope of the present invention, is a process for selecting a DNA or RNA sequence coding for all, part or precursors of a polypeptide which is inhibin or which displays an immunological or biological activity similar to inhibin, which process comprises providing one or more DNA or RNA sequences, and determining which of said sequences hybridises with a DNA or RNA sequence known to code for a part, all or precursors of polypeptides having such an activity or providing an antiserum to inhibin or part thereof and identifying host-vector combinations that express part or all of inhibin.
The above sequence may be from natural sources, may be RNA sequences, synthetic sequences, DNA sequences from recombinant DNA molecules and combinations of such sequences.
In a preferred form of the present invention the process used to identify and characterise DNA coding for at least a portion of the protein inhibin involves the extraction of mRNA species from inhibin-producing cells, their conversion to double stranded DNA (complementary DNA or cDNA) and the insertion of these into an autonomously replicating factor, such as a plasmid. This is followed by transformation of a host cell such as a bacterial strain with the factor and screening of the library produced with synthetic DNA probes which are complementary to inhibin-like mRNA or DNA sequences in order to detect those clones which contain DNA coding for inhibin as opposed to any other cell protein.
Therefore, in another form the present invention provides synthetic polynucleotide probes for the identification of inhibin-like mRNa or DNA which probes comprise a polynucleotide and a label, the said polynucleotide having a sequence selected from:
Preferably the label is a 32PO4 group attached to the 5xe2x80x2 end, or other label.
In a further embodiment the invention provides recombinant DNA molecules characterised by a DNA insert comprising a first DNA sequence which codes for amino acid sequences of all, part or precursors of inhibin or a DNA sequence which hybridises with said first sequence, said sequences being derived from whatever source including natural, synthetic, biosynthetic or semisynthetic sources and which sequences include those related by mutation, single or multiple base substitutions, deletions, insertions and inversions and including sequences which code for all, part or precursors, analogues or homologues of a polypeptide which is inhibin or which displays similar immunological or biological activity to inhibin.
Preferred recombinant DNA molecules of the invention include an expression control sequence operatively linked to said DNA insert. In one preferred form of the invention, the said DNA insert is operatively linked to the xcex2-galactosidase gene of E.coli, Other preferred control systems include those of the tryptophan (trp) operon, the leftward promoter of bacteriophage xcex (PL) and hybrid promoters such as tac or viral promoters such as that of the long terminal repeat of Moloney leukaemia virus.
A preferred recombinant DNA molecule of the present invention is a plasmid which contains a DNA insert as described above. Preferred plasmids of the present invention will be described in detail hereinafter and include pBTA22, pBTA23, pBTA28, pBTA29, pBTA30, pBTA290 and pBTA292-pBTA305.
Alternatively, said recombinant DNA molecules may comprise said DNA insert linked to DNA of a suitable bacteriophage such as bacteriophage xcex or to DNA of a virus capable of replicating in an eukaryotic cell in vitro or in whole organisms.
The invention also provides a fused gene comprising a promoter, a translation start signal, and a first DNA sequence which corresponds to or, on expression, codes for an amino acid sequence of all, part or precursors of a polypeptide which is inhibin or which has similar immunological or biological activity to inhibin, a DNA sequence which hybridises with said first sequence or a DNA sequence related by mutation, single or multiple base substitution, deletions, insertions and inversions to said first DNA sequence.
Preferred recombinant DNA molecules of the present invention comprise a plasmid into which has been inserted a DNA sequence comprising DNA of the present invention. Suitable plasmids include pBR322, pUR290, pUR291 or pUR292 or pBTA286, pUC7, pUC8 or pUC9 or pUC13 or ptrpL1 or pWT111, pWT121 or pWT131 and derivatives thereof. Also included are viral vectors such as pZIP Neo SV(X)1, vaccinia viruses, baculoviruses and derivatives thereof.
Also embraced within the present invention is a process for the manufacture of a recombinant DNA molecule, which process comprises providing a DNA insert comprising first DNA sequence which corresponds to or, upon expression codes for an amino acid sequence of all, part, analogues, homologues or precursors of a polypeptide which is inhibin or which has similar immunological or biological activity to inhibin, a DNA sequence which hybridises with said first sequence or a sequence related by mutation, single or multiple base substitutions, deletions, insertions and inversions to said first DNA sequence or hybridising sequence; introducing into a cloning vehicle said DNA insert.
Preferably said DNA sequence is introduced into the cloning vehicle in correct spacing and correct reading frame is essential reading frame with an expression control sequence.
In a further embodiment of the present invention there is provided a host transformed with at least one recombinant DNA molecule of the present invention and capable of expressing all, part or parts, or precursors of the polypeptide inhibin or a polypeptide having similar immunological or biological activity to inhibin.
Suitable hosts include bacterial cells, bacteriophages, yeasts, other fungi, vertebrate cells or insect cells, plant cells including human cells, human tissue cells or whole eukaryotic organisms.
Suitable bacterial hosts include E.coli and other enteric organisms, Pseudomonas and Bacillus. Preferred host cultures are identified as: E. coli BTA545, BTA634, BTA637, BTA647 and BTA652 and host-vector combinations as: ATCC67054-ATCC67059 and BTA1361.
Also included within the scope of the present invention is a process for transforming a host which process comprises: providing a suitable host, introducing into said host a recombinant DNA molecule of the present invention in correct reading frame.
The invention further provides expression products of the transformed host of the present invention which products comprise all, part or precursors are of a polypeptide which is inhibin or a polypeptide having similar immunological or biological properties of inhibin. Preferably these expression products are provided in substantially pure form.
In a preferred embodiment of the present invention, the expression products comprise a first polypeptide sequence homologous to the host and a second polypeptide sequence which is the amino acid sequence coding for all, part or precursors, analogous or homologues of a polypeptide which is inhibin or which has similar immunological or biological properties to inhibin.
In a preferred embodiment of the present invention, the first amino acid sequence is part or all of xcex2-galactosidase and the host cell is E.coli. In a further preferred embodiment of the invention, the first sequence is the NH2-terminal sequence of the expression product.
In a further embodiment of the present invention there is provided a process for the biosynthesis of a polypeptide which comprises all, part or precursors of inhibin or a polypeptide having similar immunological or biological activity, which process comprises: tranforming a suitable host with a recombinant DNA molecule of the present invention so that the host is capable of expressing a proteinaceous product which includes a polypeptide which is all, part of a precursor of inhibin or a polypeptide having similar biological or immunological activity; culturing said host to obtain said expression; and collecting said polypeptide.
In a preferred embodiment, the polynucleotide sequence codes on expression for a polypeptide selected from the group consisting of xe2x88x9260 to 166 amino acid sequence of FIG. 5, the xe2x88x9210 to 166 amino acid sequence of FIG. 5, the 1 to 166 amino acid sequence of FIG. 5, the xe2x88x92172 to 115 amino acid sequence of FIG. 6, the 1 to 116 amino acid sequence of FIG. 6, the xe2x88x92172 to xe2x88x921 amino acid sequence of FIG. 6, and the xe2x88x92145 to xe2x88x921 amino acid sequence of FIG. 6.
In a preferred form the expression product is formed as an insoluble inclusion body and is purified from cell extracts by centrifugation away from the soluble cell proteins. Preferred purification methods include the addition of proteolysis inhibitors and membrane disrupting agents and density gradient centrifugation. If necessary, the purified inclusion bodies may be solubilised and the proteins released by further treated, such as by selective cleavage and/or by additional purification, so as to remove unnecessary proteinaceous matter.
Furthermore, the present invention will yield an inhibin-like protein expressed by bacteria transformed with the plasmids pBTA28, pBTA29, pBTA292 and pBTA296-pBTA305.
In a further embodiment the invention provides a pharmaceutical composition comprising one or more expressed products of the invention in pharmaceutically acceptable form or synthetic equivalent.
In a further form, the invention embraces synthetic peptides which are part of inhibin and which may be inhibin agonists, antagonists or capable of eliciting an antigenic response and used to affect FSH levels or reproductive physiology.
Compositions include those suitable for oral administration or in injectable form and preferably include a pharmaceutically acceptable adjuvant. Also included in the pharmaceutical compositions of the present invention are those in sustained release form, particularly suited for implantation and sustained release in an vertibrate. In such a form the composition can be implanted into an vertibrate to affect gonadal function and removed when the desired effect is obtained.
In a further form the invention provides a vaccine comprising one or more of the expressed proteins in pharmaceutically acceptable form.
The invention also includes a method of affecting gonadal function in a vertebrate comprising administering to said vertibrate an effective amount of a pharmaceutical composition of the present invention.
In a further form the invention embraces antibody preparations prepared as a result of immunological challenge to a vertibrate by administration of one or more expression products of the present invention or pharmaceutical compositions of the present invention. Such antibody preparations include polyclonal and monoclonal antibody preparations.
Throughout this specification and claims use of the term xe2x80x9cinhibinxe2x80x9d is non-species specific and accordingly embraces related species of inhibin such as bovine, human, ovine, porcine, chicken and fish and particularly human and bovine inhibin. Also the term embraces non-glycosylated and glycosylated inhibin species.
The term xe2x80x9cvertebratexe2x80x9d embraces species of fish, amphibians, reptiles, birds and maammals including humans.
Subunit structure of inhibin
As indicated throughout the specification, inhibin as secreted into follicular fluid is a 58 kD protein comprising two subunits: a 43 kD and a 15 kD subunitxe2x80x94A and B respectively. The A or 43 kD species is a homologous protein between species with certain differences such that it would be likely to act as an antigen when one inhibin species was administered to another species. In this way it is thought that different species of inhibins could be used to raise anti-inhibin antibodies with uses hereinafter discussed.
By comparison, the B subunit or 15 kD subunit is virtually identical in amino acid sequence between a number of species, such as human and bovine species.
In certain circumstances the whole protein is cleaved to a 31 kD form comprising two subunits of molecular weight 20 kD and 15 kDxe2x80x94AC and B respectively. The 31 kD form exhibits inhibin activity and forms part of the present invention. Cleavage of the 58 kD form to the 31 kD form liberates a polypeptide fragment referred to as the AN fragment which itself could play a significant role in regulating gonadal function and thus is also part of the present invention.